1. Field of Invention
This invention relates to hemoglobin measurement of blood samples, and more specifically relates to methods of correction of particle interference to hemoglobin measurement on automated hematology analyzers.
2. Background
Determination of total hemoglobin concentration is indicative of the oxygen-carrying capacity of whole blood. More than 300 abnormal hemoglobins have been discovered upon examination of patients with clinical symptoms and by electrophoretic surveys of a clinically normal population. Many of these abnormalities result in clinical pathologies having altered hemoglobin levels or hemoglobin having an altered ability to bind oxygen.
Commercial hematology analyzers are equipped to measure and report hemoglobin concentration of blood samples. On most hematology analyzers, the process of measuring hemoglobin concentration of a blood sample involves lysing red blood cells in a blood sample and forming a hemoglobin chromogen in the sample mixture, measuring spectrophotometric absorbance of the sample mixture at a predetermined wavelength, and reporting a total hemoglobin concentration of the blood sample.
In the sample mixture used for measuring hemoglobin concentration, red blood cells are lysed to release hemoglobin molecules, other cellular particles such as white blood cells, nucleated red blood cells and platelets remain in the sample mixture, although they may be damaged or shrunk in size. It is known that these cellular particles present in the sample mixture interfere with the spectrophotometric measurement of hemoglobin, because the cellular particles absorb and scatter the incident light used in the measurement. In normal blood samples, white blood cell concentration is typically from 4×103/μL to 9×103/μL and platelet concentration is typically from 200×103/μL to 400×103/μL. However, in some pathological conditions, for example, severe lipemia or proteinemia, white blood cell concentration can be higher than 20×103/μL and platelet concentration can be higher than 700×103/μL. In some leukocytosis samples, the white blood cell concentration can be substantially higher than 100×103/μL. These cellular particles in the sample mixture used for hemoglobin measurement may cause significant overestimation of the hemoglobin concentration. The particle interference has also been encountered with blood samples with a significant amount of hemoglobin S and severe microcytosis/hypochromasia.
Several methods are currently used to prevent, or correct the interference of cellular particles to hemoglobin measurement. In the Clinical and Laboratory Standards Institute (formerly NCCLS) manual reference method, when the absorbance of the sample mixture at 750 nm is ≧0.003, or when the absorbance ratio (absorbance at 540 nm vs. absorbance at 504 nm) is <1.59, it is required to filter the sample mixture through a 0.22 μm filter to remove the particles.
A common practice in clinical diagnosis laboratories, as often recommended by hematology analyzer manufacturers, after the hematology analyzer reports abnormally high white blood cell concentration, the blood sample is analyzed again with a pre-dilution to determine hemoglobin concentration. In this process, the blood sample is diluted by a saline solution, or diluted by a diluent used on the hematology analyzer to reduce the concentration of the particles, for example, to reduce the white blood cell concentration down to below 20×103/μL or a level that the manufacturer recommends. Then, the pre-diluted sample is analyzed again on the instrument to measure hemoglobin. The obtained hemoglobin concentration from the pre-diluted sample is used to calculate the hemoglobin concentration of the blood sample, taking account for the dilution. This method has several disadvantages. The blood sample has to be analyzed twice on the instrument. It is time consuming and requires manual preparation by an operator. The pre-dilution introduces additional errors originated from pipetting the blood and the diluent, and manually mixing the blood prior to and after the dilution. Moreover, for a blood sample that has a low hemoglobin concentration, further dilution reduces accuracy of hemoglobin measurement.
Another known method to correct particle interference to hemoglobin measurement is to measure the absorbance of the sample mixture at two different wavelengths. The first wavelength is in the visible region, for example at 540 nm, which is used to measure the hemoglobin chromogen. The second wavelength is at infra-red region, for example at 800 nm to measure turbidity caused by the cellular particles. The absorbance at the second wavelength is used to correct the measurement at the first wavelength to produce a corrected hemoglobin concentration. A hand-held device using this method is commercially available from HemoCue AB (Ängelholm, Sweden) under the trade name of Hemocue Hb 201+. This device is used for measuring hemoglobin only, not other hematology parameters. In some hematology laboratories, particularly in Europe, after a blood sample has been analyzed on a hematology analyzer and reported to have an abnormally high white blood cell concentration, Hemocue Hb 201+ is then used to measure and report hemoglobin concentration.
Accordingly, there is a need for a method, particularly an automated process on automated hematology analyzers, to correct the interference of cellular particles to the measurement of hemoglobin concentration.